The long-term objective is to understand the physical mechanism of DNA condensation by multivalent cations, so as to comprehend the factors controlling compaction in viruses and cells and in preparative and genetic engineering applications. Understanding DNA condensation has two main aspects: equilibrium and kinetic characterization of the condensation process, and elucidation of the forces underlying condensation. DNA condensation seems to involve four major stages: (l) initial rapid association of a few chains, (2) collapse of this complex at some critical size, (3) growth of the critical nucleus by accretion, and (4) long-time aggregation of fully-formed condensed particles to produce larger particles. Each stage is reversible, but the process as a whole seems to be nucleation-limited. The major energetic contributions to DNA condensation are electrostatics, hydration, and distortions of the double helix. In the previous grant proposal we emphasized the role of helix distortion. We have accumulated significant evidence supporting this point of view, particularly with divalent cations, but feel that the evidence implicating hydration forces and electrostatics is still very strong. Specific aims are: * Perform early-time light scattering and EM experiments to characterize the stages leading to formation and collapse of the critical nucleus. * Combine early-time data with light scattering and electron microscopic data on later stages, to obtain a consistent set of rate and equilibrium constants for all stages of the condensation process. * Use cluster aggregation ideas to characterize the fractal dimensions of the large aggregates formed in the last stage, to determine whether they are formed in a diffusion- or reaction-limited process. * Study the effects of solution variables - multivalent cations, salt, cosolvents, temperature, and osmotic pressure - on the equilibrium steps in DNA condensation and the morphologies of the resulting structures. * Continue development of a model of complementary charge patterning of counterions on the DNA surface, as the basis for an electrostatic explanation of attractive forces between DNA molecules. * Collaborate on osmotic stress measurements to develop a library of hydration force-distance parameters for condensing agents as functions of their size and charge. * Investigate the role of helix perturbations by chemical and enzymatic probing of supercoiled plasmids with Z-DNA-forming insertion sequences, and Raman and CD spectroscopy of cation-DNA systems. * Use force measurements on single, stretched-out DNA molecules to ascertain whether charge neutralization causes "crumpling", or whether side-by-side attractive forces are required to initiate condensation.